Você está na página 1de 8

BEST: International Journal of Humanities, Arts, Medicine and Sciences (BEST: IJHAMS) Vol.

1, Issue 2, Nov 2013, 15-22 BEST Journals


Department of Biology, College of Science, Al Muthanna University, Al Muthanna, Samawah, Iraq


Department of Biology, College of Science, University of Babylon, Babylon, Iraq

A total of 294 stool samples were collected from patient children, their ages were between 1 month to 6 years and suffering from diarrhea disease during period December 2012 and February 2013from Al-Muthana public health laboratory in Muthana province - Iraq. All suspected isolates were screening by traditionally tests and then confirmed by Vitek 2 system and PCR technique(16S r RNA gene).The results showed that; there were 12 (4.08%) positive isolates of Aeromonas hydrophila. The SDS-PAGE method was used to analysis outer membrane proteins (OMPs) profile. The result revealed that, the OMPs molecular weight was around 44 KDa.

KEYWORDS: Identification A. hydrophila, Isolation of OMP, SDS-PAGE Analysis INTRODUCTION

Diarrhea is a leading cause of childhood mortality and morbidity in developing countries and ranks among the most common causes of disease in children worldwide. Among bacterial etiologies of diarrhea, A. hydrophila is recognized increasingly as a clinically significant enteric pathogen. However, there are limited data on the prevalence and associated severity of diarrheal disease caused by A. hydrophila in many regions [1]Moreover a strong association between gastroenteritis and Aeromonas species has been shown in children, adults who are older than 60 years and in cases of travelers diarrhea[2] In fact, the three most common human infections caused by Aeromonas species are gastrointestinal infection, skin and soft-tissue infection, and bacteremia in immunocompromised individuals[3]Virulence factors of A. hydrophilaare present in two forms, cell-associated structures, and extracellular products. Among the cell-associated structures are pili, flagella, outer membrane proteins, lipopolysaccharide, and capsules. The major extracellular products include cytotoxic, cytolytic, hemolytic, and enterotoxic proteins [4] The outer membrane of Gram-negative pathogenic bacteria has an important role in the interaction with hosts in the bacterial pathogenicity during adherence, uptake of nutrients from the host and eliminating host-defense mechanisms [5] In previous study, methods based on restriction patterns of the polymerase chain reaction (PCR) amplified 16s rRNA genes were used for the identification of clinical strains of Aeromonas spp. Molecular techniques such as PCR and outer membrane based immunoassay have been used for detection and or identification of Aeromonas spp from food or environmental or clinical samples [6]. Consequently, and from the above brief discussion A. hydrophila starts to have more space and attention from both Iraqi scientists and researchers alike. Therefore the current studyincluded isolation and identification of A.hydrophilafrom diarrhea collected samples, isolation and SDS-PAGE analysis of outer membrane protein (OMP).


Naseem Q N. Dubai & Azhar A. L Al-Thahab


Samples Collection A total of 294 stool samples were collected between December 2012 and February 2013 from patient children their ages were between (1month_6years) and suffering from diarrhea disease. These stool samples were obtained from Al-Muthana public health laboratory.

Cultural Methods All samples were activated in APW media at 37 0C for 18-24h, and growing on culture media which are TCBS, MacCon key and Blood agar at 37 0C for 18-24hr [7]. Biochemical Identification To confirm initial diagnosis of bacteria a manual biochemical tests were used such as catalase, oxidase, Indole, methyl red, simmone citrate, gelatin liquefaction and vogues-proskauer test[8]. Vitek System Identification The Vitek 2 system assay has been used to confirm identification of A.hydrophila. This system performed according to the manufacturer's instructions (Biomerieux Company, France). Molecular Identification A polymerase chain reaction (PCR) technique was used to identify A.hydrophilaby amplify genes of 16Sr RNA gene from genomic DNA. DNA extraction from Gram negative bacteria was performed according to the genomic DNA purification kit supplemented by the manufacturing company (Geneaid/Taiwan). Gel electrophoresis has been used for detection of DNA by UV transilluminator[9]. The primers selection according to [8],[10] recommendations and used for diagnosis A.hydrophila. These primers synthesized by AccuOligo- Bioner Company, Korea, as shown in table (1). Table 1: The Sequence of Forward and Reverse Primers Primer Type Forward 16Sr RNA - F Reverse 16Sr RNA - R Primer Sequence 5-CCAGCAGCCGCGGTAATACG-3 5-TACCAGGGTATCTAATCC-3 Product Size 300 bp

PCR Mixture solution was according to information of manufacturing company(Master mix, Geneaid/Taiwan) and PCR Program conditions was listed in table (2)[8]. Ten lstandard molecular weight of DNA ladder (marker) was loaded in first well on 1% agarose gel and each well has been loaded with 10l of PCR product (DNA sample). Electrophoresis runs at 80 volt/cm for 1hr. Table 2: Amplification Conditions Steps Initial denaturation Denaturation Annealing Elongation Final elongation Isolation of Outer Membrane Protein (OMP) The OMPs of A. hydrophila were prepared according to the method of [11] with few modifications. Temperature 94 oC 94 oC 52 oC 72 oC 72 oC Time 3 min 30 sec 30 sec 30 sec 10 min No. of Cycles

30 cycle

Isolation of Outer Membrane Protein of Aeromonas hydrophila Recovered from Children with Diarrhea


From 2-3 litter of brain heart infusion broth was inoculated by the most virulence A. hydrophila isolate and incubated for 24- 48 h at 37 C in a shaker incubator. After incubation period the bacteria was harvested by centrifugation at 6000 rpm for 15 min. The obtained pellet was washed twice by 40 ml of phosphate buffer saline (PBS, pH 7.2) and once in 40 ml of Hepes buffer (pH 7.4) then centrifuged at10000 rpm for 10 min. The cells were re suspended in 20 ml Hepes buffer and disrupted by sonication for 30 min at 10 watt an interval of 30 second with ice .Unbroken cells and cellular debris were removed by centrifugation at 4000rpm for 15 min at 4oC.The resultant supernatant was then further centrifuged at 10000rpm for 1 hr at 4oC.The supernatant was discarded and the pellet suspended in 20 ml of 2% triton X-100 and then incubated at room temperature for 30 min to solubilize the inner membrane .The suspension was then centrifuged at 10000 rpm for 1 hr at 4oC. The supernatant was discarded and the pellet suspended in 1 ml of PBS (pH 7.2) and stored at -20oC until use .Protein concentration of the OMP preparation was estimated by the Biuret method [12] SDS-Page Analysis of Outer Membrane Proteins (OMPS) The OMP analyzed by SDS-page was prepared according to the method of[13] using 12.5% (w/v) polyacrylamide in the resolving gel with few modifications. The OMP sample were diluted with sample buffer in ratio of 4:1 and heated at 95 C for 5 min.30 l of the OMP sample containing 200 g of protein was loaded in each lane of the gel. The gel was run at 150v for 6 hrs, and then stained with coomassie brilliant blue R-250 staining solution for overnight and then distained with distaining solution. The molecular weight (MW) of the protein was calculated by extrapolation of relative mobility of the unknown samples against that of standard molecular weight markers. SDS-PAGE analysis reagents are shown in table (3). Table 3: SDS-Page Solution Solution Components of Solution Tris. Distilled water.Adjustment of pH 8.8 with concentrated HCl and 100 ml volume was made with distilled water. Tris. Distilled water.Adjustment of pH 6.8 with concentrated HCl and 100 ml volume was made with distilled water. Tris Glycine SDS Distilled water to make 1 M TrisHCl (pH 6.8) 50% Glycerol 10% SDS (w/v) 1% Bromophenol blue Distilled water Acrylamide Bisacrylamide Distilled water to make 100 ml Polyacrylamide solution 1M TrisHCl (pH 8.8) Distilled water 10% SDS TEMED 10% Ammonium per sulphate (w/v) Amount 12.1g 50ml 12.1g 50ml 3.025 g 14.413 g 1.0 g 1000 ml 0.6 ml 5 ml 2 ml 1 ml 0.9 ml 29.2 g 0.8 g 100 ml 12.5 ml 11.2 ml 6.2 ml 0.3 ml 20 l 100 l

1. 1M TrisHCl (pH 8.8)

2. 1M TrisHCl (pH 6.8)

3. Electrophoresis buffer (pH 8.3)

4. Sample buffer (5X)

5.polyacrylamide solution

6. Running Gel (12.5%)


Naseem Q N. Dubai & Azhar A. L Al-Thahab

7.Staking Gel

8.Staining solution

9.Distaining solution

Table 3: Contd., Ployacrylamide solution 1M TrisHCl (pH 6.8) Distilled water 10% SDS TEMED 10% Ammonium per sulphate (w/v) Coomassie brilliant blue Methanol Acetic acid Distilled water Methanol Acetic acid Distilled water

1.67 ml 1.25 ml 7.03 ml 0.4 ml 10 l 50 l 0.15% in dark bottle 45% 10% 45% 45% 10% 45%


Isolation of A. hydrophila Two hundred ninety four of diarrheic stool samples were collected from Al-Muthana public health laboratory. The results showed that, there were 12 (4.08%) positive isolates of A.hydrophila (Figure 1). These results are almost agree with [14] were the highest prevalence of Aeromonas was observed in infants (< 1 years old) in Brazil. As well as, several authors reported that the high frequency of Aeromonas was found in infants and elder [15]. Moreover, [16] found out diarrhea disease that caused by Aeromonas was more frequently in children whom age between 1-3 years, in South India. Likewise, [17] reported that from one hundred twenty eight (128) diarrheic stool samples analyzed, 4 (3. 12%) were found to be positive for Aeromonas hydrophila in Nigeria.

Figure 1: Frequency of Aeromonas hydrophila from Diarrheicstool Samples According to Children Patients Age Identification of A. Hydrophila Aeromonas hydrophilashowed a yellow shine colour on TCBS agar, pale (non -lactose fermenters) on the MacConkey agar, smooth, convex, rounded, -hemolytic colonies and pale white to grey colour on blood agar[8]. In terms of, initial biochemical tests A. hydrophila showed a positive result to each of catalase, oxidase, Indole, methyl red, simmone citrateand gelatin liquefaction. The current results of the biochemical tests in this study are almost finding in the other researchers reports [18],[19] While, A. hydrophila was gave variable results tovogues-proskauer. Vitek 2 system is an efficient biochemical testto confirm identification of A. hydrophila [19].The analytical profile index of this system has showed probability identification between (97%-99%) percentage. In this study a polymerase chain reaction (PCR) technique was used toidentify A.hydrophilaby amplify genes of 16Sr RNA gene from genomic DNA of all A.hydrophila isolates. All isolates have given a positive results for 16Sr RNA

Isolation of Outer Membrane Protein of Aeromonas hydrophila Recovered from Children with Diarrhea


(300)bp. The resultsillustrated in (Figure 2). In fact, [8] stated thatmost of A. hydrophila isolates shown a positive result to detected for (16SrRNA) gene. In addition to that, they reported the ribosomal mainly 16Sr RNA gene has confirmed to be a stable and specific molecular marker for the identification of A. hydrophila bacteria.

Figure 2: Agarose Gel Electrophoresis of PCR Amplified of 16S rRNA Gene 300 bp of Aeromonas hydrophila Isolates for 1 hr at 80 Volt. Lane 1 DNA Marker (100bp Ladder). Lane 2,3,4,5,6,7,8,9,10,11,12,13 Amplify of 16Sr RNA Gene in A. Hydrophila Separation and SDS-Page Analysis of OMP from A. hydrophila The outer membrane (OMP) play an important role in reaction with hosts in the bacterial pathogenicity throughout adherence, parasite on the host and eliminating host-defense mechanisms[6]. Indeed, in this study OMP A. hydrophila was separated from the most virulence isolate. As well as, the protein concentration was estimated and gave 2.2 g\L. Moreover, SDS- page analysis of the OMP of A. hydrophila revealed that the molecular weight (MW) of the polypeptide band estimated by comparison with standard MW markers run parallel inrange 28-180 KDa, and the OMP was revealed 44KDa molecular weight (Figure 3).

Figure 3: For 6 hr at 150 Volt. Lane 1: Protein Marker (180KDa Ladder). Lane 2: OMP 44KDa A. hydrophila

Different researchers noticed that major protein bands in the range of 30 and 45 kDa [20] and this finding is agree with the observe result of this study. As well as, likewise [21] reported that major proteins also ranged between 55 and 28 kDa. Moreover, in the study of OMP highly virulence group of strains, [22] indicated that major proteins of MW of 30 kDa and proteins are predominant. In fact, these variations are not unexpected due to the complex and diverse nature of Aeromonas spp. [20]. Therefore, further studies in Iraq need to be done on the area of investigate of OMP antigen ability to induce immunity response and make sure if it can be used as a potential vaccine to control A. hydrophila.


Naseem Q N. Dubai & Azhar A. L Al-Thahab

1. Mansour, A. M., Elkhalek, R. A., ShaheenH. I., Mohammady, H.E., Refaey, S., Hassan,K., Riddle,

M., Sanders, J. W., Sebeny,P. J., Young, S. Y. N. &Frenck, R. 2012. Burden of Aeromonas hydrophila associated diarrhea among children younger than 2 years in rural Egyptian community. Journal Infection DevCtries 6(12): 842-846. 2. Sinha, S., Shimada, T., Ramamurthy, T., Bhattacharya, S.K., Yamasaki, S., Takeda, Y. & Balakrish, G.N. 2004. Prevalence, serotype distribution, antibiotic susceptibility and genetic profiles of mesophilic Aeromonas species isolated from hospitalized diarrhoeal cases in Kolkata, India. Journal of Medical Microbiology 53: 527-534. 3. Hochedez, P., Hope-Rapp, E., Olive, C., Nicolas, N., Beaucaire,G. &Cabi,C. 2010. Bacteremia Caused by Aeromonas hydrophila Complex in the Caribbean Islands of Martinique and Guadeloupe. The American Society of Tropical Medicine and Hygiene 83(5):11231127. 4. EPA, Environmental Protection Agency. 2006. Aeromonas: Human Health Criteria Document. EPA, Washington, DC.USA. Available at:http://water.epa.gov/action/advisories/drinking/upload/2009_02_03_criteria_humanhealth_microbial_aeromon as-200603.pdf. [Accessed 2/8/2013]. 5. 6. Seltman, G. & Holst, O. 2000. The Bacterial Cell Wall. Berlin, Heidelberg and New York: Springer-Verlag. Maiti, B., Raghunath, P., Karunasagar, I. & Karunasagar, I. 2009. Cloning and expression of an outer membrane protein OmpW of Aeromonas hydrophila and study of its distribution in Aeromonas spp. Journal of Applied Microbiology 107:11571167. 7. AL-Fatlawy, H. N. K. & AL-Ammar M. H. 2013. Molecular study of Aeromonas hydrophila isolated from stool samples in Najaf (Iraq). International Journal Microbiology Research Vol, 5, Issue 1, pp. 363-366. 8. MaccFadin, J. K. 2000. Biochemical test for identification of medical bacteria. (3rd ed .). Lippincott Williams and Winkins .AwolterKlumerCompany. Philadelphia Baltimor. New York. 9. Sambrook, J. &Rusell, D.W. 2001. Molecular cloning.A laboratory manual. 3rd ed. Cold Spring Harbor Laboratory Press, NewYork, USA. 10. Yogananth, N., Bhakyaraj, R., Chantchuru, A., Anbalagan,T. &MullaiNila, K. 2009. Detection of virulencegene in Aeromonas hydrophila isolated from fish samples using PCR technique. Global Journal Biotechnology Biochemical 4: 5153. 11. Behera, T., Nanda, P. K., Mohanty, C., Mohapatra, D., Swaina,P., Das, P. K., Routray, P., Mishra, B. K. & Sahoo, S.K. 2010. Parenteral immunization of fish, Labeorohita with Poly D, L-lactide-co-glycolic acid (PLGA) encapsulated antigen microparticles promotes innate and adaptive immune responses. Fish & Shellfish Immunology 28: 320-325. 12. Tietz, N. W., Burtis, C. A., Ashwood, E.R. & Saunders, W.B. 1999. Text Book of Clinical Chemistry. 3rd Ed. 477-530.

Isolation of Outer Membrane Protein of Aeromonas hydrophila Recovered from Children with Diarrhea


13. Sachan, N., Agarwal, P.K. &Singh,V. P. 2012. Study on Outer Membrane Protein (OMP) Profile of Aeromonas Strains using SDS-PAGE. Research.Vet World Vol.5 (3): 173-177. 14. Guerra, I. M. F., Fadanelli, R., Figueir, M, Schreiner, F., Delamare, A. P. L. Wollheim, C., Costa, S. P. O. & Echeverrigaray, S. 2007. Aeromonas associated diarrhoeal disease in south brazll: prevalence virulence factors and antimicrobial resistance. Brazilian Journal Microbiology 38: 638-643. 15. Janda, J.M. & Abbott, S.L. 2010. The genus Aeromonas, taxonomy, pathogenicity, and infection. Clinical Microbiology Reviews 23: pp. 35-73. 16. Meiyanti, S., Salim, O. C., Surjawidjaja, J. E. &Lesmana, M. 2010. Isolation and antibiotic sensitivity of Aeromonas from children with diarrhea. Universal Medicina Vol, 29. No.1. 17. Rogo, L.D., Attah, A., Bawa, E., Aliyu, A.M., Aliyu, M.S. &Gaiya, A.Z. 2009. Antimicrobial susceptibility pattern of Aeromoas Hydrophila among patients presented with diarrheal attending two teaching hospitals in northern Nigeria. Bayero Journal of Pure and Applied Sciences 2(1): 91 95. 18. Erdem, B., Kariptas, E., Cil, E., Isik, K. 2011. Biochemical identification and numerical taxonomy of Aeromonas spp. isolated from food samples in Turkey. Turkey Journal Biology 3 463-472. 19. Kivanc, M., Yilmaz, M., Demir, F. 2011. The occurrence of Aeromonas in drinking water and the Porsuk River. Brazilian Journal of Microbiology 42: 126-131. 20. Kuijper, E. J., Van-Alphen, L., Leenders E. &Zanen, H. C. 1989. Typing of Aeromonas strains by DNA restriction endonuclease analysis and polyacrylamide gel electrophoresis of cell envelops. Journal Clinical Microbiology 27: 1280-1285. 21. Santos, Y., Bandin I. &Toranzo, A. E. 1996. Immunological analysis of extracellular products and cell surface components of motile Aeromonas isolated from fish. Journal Applied Bacteriology 81: 585-593. 22. Dooley, J. G. S. & Trust, T. J. 1988. Surface protein composition of Aeromonas hydrophila strains virulent for fish: Identification of a surface array protein. Journal Bacterioogy 170: 499-506